Synthesis and antibacterial activity of novel phosphonium salts on the basis of pyridoxine

Article abstract of DOI:10.1016/j.bmc.2013.04.051

A series of 13 phosphonium salts on the basis of pyridoxine derivatives were synthesized and their antibacterial activity against clinically relevant strains was tested in vitro. All compounds were almost inactive against gram-negative bacteria and exhibited structure-dependent activity against gram-positive bacteria. A crucial role of ketal protection group in phosphonium salts for their antibacterial properties was demonstrated. Among synthesized compounds 5,6-bis[triphenylphosphonio(methyl)]-2,2,8-trimethyl-4H-[1,3] dioxino[4,5-c]pyridine dichloride (compound 20) was found to be the most effective towards Staphylococcus aureus and Staphylococcus epidermidis strains (MIC 5 μg/ml). The mechanism of antibacterial activity of this compound probably involves cell penetration and interaction with genomic and plasmid DNA.

Full text of DOI:10.1016/j.bmc.2013.04.051

Bioorganic & Medicinal Chemistry 21 (2013) 4388–4395
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antibacterial activity of novel phosphonium salts
on the basis of pyridoxine
Mikhail V. Pugachev a, Nikita V. Shtyrlin a, Lubov P. Sysoeva a, Elena V. Nikitina a, Timur I. Abdullin a,
Alfiya G. Iksanova a, Alina A. Ilaeva a, Rashid Z. Musin b, Eugeny A. Berdnikov a, Yurii G. Shtyrlin a,
⇑
a Research and Educational Center of Pharmacy, Kazan Federal University, Kazan 420008, Russian Federation
b A.E. Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Centre of the Russian Academy of Sciences, Kazan 420088, Russian Federation
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 13 phosphonium salts on the basis of pyridoxine derivatives were synthesized and their anti-
bacterial activity against clinically relevant strains was tested in vitro. All compounds were almost inac-
tive against gram-negative bacteria and exhibited structure-dependent activity against gram-positive
bacteria. A crucial role of ketal protection group in phosphonium salts for their antibacterial properties
was demonstrated. Among synthesized compounds 5,6-bis[triphenylphosphonio(methyl)]-2,2,8-tri-
methyl-4H-[1,3]dioxino[4,5-c]pyridine dichloride (compound 20) was found to be the most effective
towards Staphylococcus aureus and Staphylococcus epidermidis strains (MIC 5 lg/ml). The mechanism of
antibacterial activity of this compound probably involves cell penetration and interaction with genomic
and plasmid DNA.
Received 2 March 2013
Revised 12 April 2013
Accepted 19 April 2013
Available online 29 April 2013
Keywords:
Quaternary phosphonium salts
Pyridoxine
Antibacterial activity
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
tetradecyl triphenyl phosphonium bromide (TTP) functionalized
few-layered graphite (FG)24 exerted long-term antimicrobial activ-
Organophosphorus compounds have been extensively utilized
in organic synthesis. Among them quaternary phosphonium salts
are of special interest due to their use in material sciences1–10
and medicinal chemistry.11–17 In particular, these compounds have
been applied as intracellular antioxidants,11–13 anticholinesterase
inhibitors,14 tumor imaging agents15,16 and chemotherapeutic
agents.17
ity against E. coli and S. aureus.
Chemical modification of biologically active compounds of nat-
ural origin is one of the most efficient approaches in drug develop-
ment. Among them, vitamin B6 (pyridoxine) is of particular interest
as a starting compound as it participates in over a hundred enzy-
matic reactions involved in biosynthesis, metabolism, and regula-
tory functions in living organisms.25–29
Antimicrobial properties of some quaternary phosphonium
salts have been reported. Phosphonium salts grafted on styrene–
divinylbenzene copolymers were shown to exhibit antibacterial
activity against Staphylococcus aureus, Escherichia coli and Pseudo-
monas aeruginosa.18 Triphenylphosphonium-modified PPO (poly-
phenylene oxide) polymer also found to have antimicrobial
activity on Staphylococcus epidermidis and Escherichia coli.19 Tala-
driz et al., recently demonstrated antitrypanosomal activity of ben-
zyltriphenylphosphonium salts against Trypanosoma brucei.20
Benzophenone-derived bisphosphonium salts exhibited lethal
activity towards human protozoan parasite Leishmania.21 Kanaza-
wa et al.,22 synthesized di- and trimethyl-substituted phospho-
nium salts with long alkyl chaines (C10–C18) with bacteriostatic
action against 11 widespread pathogens including methicillin-
resistant S. aureus (MRSA). Organ-clay minerals intercalated by tet-
radecyl tributyl phosphonium bromide (TDTB)23 as well as
In our study, we describe the synthesis of novel derivatives of
quaternary phosphonium salts on the basis of pyridoxine and its
6-hydroxymethyl derivatives. We reveal the effect of quantity
and location of phosphonium fragments in pyridoxine molecule
on the antibacterial activity of synthesized substances as well as
the probable mechanism of action.
2. Results and discussion
2.1. Chemistry
The synthesis of the target compounds was based on the nucle-
ophilic substitution of chlorine derivatives of pyridoxine with tri-
phenylphosphine resulting in the corresponding phosphonium
salts (Schemes 1–7).
Various known synthetic approaches were used for the synthe-
sis of different chlorine derivatives of pyridoxine, such as: intro-
duction of ketal or acetate protection to the hydroxyl groups for
selective functionalization of pyridoxine and its derivatives, the
⇑
Corresponding author. Tel./fax: +7 843 233 75 31.
E-mail address: Yurii.Shtyrlin@ksu.ru (Y.G. Shtyrlin).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.051

M. V. Pugachev et al. / Bioorg. Med. Chem. 21 (2013) 4388–4395
4389
OH
HO
HO
O
O
HO
O
O
P+Ph3
2Cl-
a, b
OH
d
Cl
P+Ph3
Cl-
c
N+
N
HCl
N
N
H
4
3
2
1
Scheme 1. Reagents and conditions: (a) (CH3)2CO, HCl, rt to 20 °C, 3 h;30 (b) CH2Cl2, SOCl2, 25 °C, 2 h;31 (c) PPh3, CH3CN, reflux, 7 h; (d) H2O, HCl, 25 °C, 24 h.
O
O
O
O
HO
HO
O
O
O
O
e
HO
AcO
c
d
AcO
AcO
a, b
OH
OH
OH
Cl
P+Ph3
Cl-
N
N
N
N
N
6
5
7
8
1
f
OH
OH
HO
P+Ph3
2Cl-
N+
H
9
Scheme 2. Reagents and conditions: (a) (CH3)2CO, HCl, 0 °C, 24 h;32 (b) H2O, H2CO, NaOH, 70 °C, 60 h;33 (c) CH2Cl2, Ac2O, Et3N, rt, 30 h; (d) CHCl3, MsCl, Et3N, 0 °C to rt, 10 h;
(e) PPh3, CH3CN, reflux, 7 h; (f) H2O, HCl, 25 °C, 24 h.
HO
O
HO
HO
HO
HO
HO
O
a-c
OH
e
d
Cl
Cl
Cl
OH
P+Ph3
OH
2Cl-
N
N
N+
H
N+
H
Cl-
11
10
1
12
Scheme 3. Reagents and conditions: (a) (CH3)2CO, HCl, 0 °C, 24 h;32 (b) H2O, H2CO, NaOH, 70 °C, 60 h;33 (c) CH2Cl2, SOCl2, rt, 4 h;34 (d) H2O, HCl, 55 °C, 72 h; (e) PPh3, CH3CN,
reflux, 7 h.
HO
HO
O
HO
HO
Ph3+P
2Cl-
O
O
a-f
O
g
h
i
OH
OH
O
O
O
O
O
O
OH
OH
OH
Ph3+P
Cl-
HO
Cl
N+
H
N
N
N
14
N
1
13
15
16
Scheme 4. Reagents and conditions: (a) (CH3)2CO, HCl, 0 °C, 24 h;32 (b) H2O, H2CO, NaOH, 70 °C, 60 h;33 (c) (CH3)2CO, HCl, rt, 20 h;35 (d) CHCl3, MCPBA, rt, 48 h;36 (e) Ac2O,
100 °C, 4 h;36 (f) CHCl3, CH3OH, CH3ONa, 0 °C to rt, 2 h;36 (g) CHCl3, MsCl, reflux, 5 h; (h) PPh3, CH3CN, reflux, 7 h; (i) H2O, HCl, 25 °C, 24 h.
HO
HO
HO
HO
O
O
a-f
g
O
O
h
P+Ph3
3Cl-
OH
Cl
P+Ph3
2Cl-
Ph3+P
Cl
Ph3+P
H3C
N
N+
H
N
N
1
17
18
19
Scheme 5. Reagents and conditions: (a) (CH3)2CO, HCl, rt to 20 °C, 3 h;30 (b) Pyr, Ac2O, rt, 30 h;37 (c) CHCl3, MCPBA, rt, 72 h;37 (d) Ac2O, 80–90 °C, 4 h;37 (e) CH3OH, CH3ONa,
0 °C, 2 h;37 (f) SOCl2, 0 °C, 0.5 h;37 (g) PPh3, CH3CN, reflux, 7 h; (h) H2O, HCl, 25 °C, 24 h.
introduction of additional hydroxymethyl group into position 6 of
the pyridine ring of pyridoxine by its hydroxymethylation in alka-
line medium, the modification of methyl group in the position 2 of
the pyridine ring of pyridoxine via the pyridine N-oxide rearrange-
ment, and chlorination of alcoholic hydroxyl groups at the last
steps of synthesis.
The preparation of some known intermediates (hydroxy and
chlorinated pyridoxine derivatives)30–37 was described in the reac-
tions: a, b (Scheme 1), a, b (Scheme 2), a–c (Scheme 3), a–f
(Scheme 4), a–f (Scheme 5), a–g (Scheme 7).
Chlorine derivatives (7, 14, 23) were obtained via chlorination
of the hydroxymethyl group of compounds (6, 13, 22) using

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M. V. Pugachev et al. / Bioorg. Med. Chem. 21 (2013) 4388–4395
and 6 positions of the pyridine ring. Its MIC value on both S. aureus
and S. epidermidis was 5 lg/ml that was twice higher than for
vancomycin.
O
O
HO
HO
O
O
b
a
Cl
Cl
P+Ph3
P+Ph3
2Cl-
P+Ph3
P+Ph3
3Cl-
N
N
N+
H
2.3. Cytotoxity of compound 20
10
20
21
We assessed cytotoxicity of phosphonium salt 20 on human
skin fibroblasts with the use of metabolic MTT-assay as described
in experimental part. The cytotoxic concentration (CC50) of com-
Scheme 6. Reagents and conditions: (a) PPh3, CH3CN, reflux, 7 h; (b) H2O, HCl,
25 °C, 24 h.
pound 20 was found at 160 lg/ml.
methanesulfonyl chloride as a chlorinating agent in the presence of
an excess of triethylamine.
2.4. DNA-damaging activity of compound 20
For the preparation of monochlorine derivative 11 the hydroly-
sis of dichloride 10 containing a six-membered ketal cycle was per-
formed in acidic conditions at 55 °C. In this reaction also took place
selective hydrolysis of the chlorine atom of the chloromethyl group
in the 2-position of pyridine ring.
The interaction of compound 20 with bacterial DNA as a prob-
able target was assessed by different assays. With the use of comet
test we studied the effect of compound 20 on DNA stability in S.
aureus cells. Cell treatment with the compound induced notable
DNA fragmentation and its migration out of cells at the concentra-
Quaternary phosphonium salts (3, 8, 12, 15, 18, 20, 24) on the
basis of pyridoxine were obtained by the treatment of chlorine
derivatives of pyridoxine with 2.0-fold molar excess of triphenyl-
phosphine in CH3CN at reflux temperature during 7 h. Unexpect-
edly, we found that the reaction of chlorine derivatives 14 with
triphenylphosphine has led not only to the formation of phospho-
nium salt 15 but also to the removal of the ketal seven-membered
cycle. In the last step, hydrolysis of the ketal protecting group un-
der acidic conditions of mono-, bis- and trisphosphonium salts was
carried out. The products of hydrolysis (4, 9, 16, 19, 21, 25) were
identified as hydrochlorides.
tion as low as 10
(Fig. 1a).
lg/ml as revealed by fluorescent microscopy
The additional agarose gel electrophoresis of genomic DNA ex-
tracted from bacteria demonstrated that the compound 20 caused
partial DNA fragmentation at concentrations 0.01–10 mg/ml
(Fig. 2). These results indicate that compound 20 may affect S. aur-
eus viability by direct of indirect damage of genomic DNA.
2.5. Effect of compound 20 on plasmid DNA state
Plasmid DNA was isolated from S. aureus treated with the com-
pound 20 (0.01–1 mg/ml) and assessed with the aid of dynamic
light scattering technique. We found that the treatment results in
marked increase of hydrodynamic diameter of plasmid DNA mole-
cules from 360.7 (control) to 500.9 and 452.3 nm (for compound
20 concentration of 0.01 and 0.1 mg/ml, respectively) (Table 2).
Zeta potential of native plasmid DNA was almost À51.9 mV at
pH 7.4. After cell exposure to compound 20 zeta potential of iso-
lated plasmid DNA markedly decreased to À46.6 and À33.2 mV
for 0.01 and 0.1 mg/ml concentrations, respectively. Such changes
in hydrodynamic diameter and zeta potential of plasmid DNA indi-
cate that compound 20 penetrates bacterial cells and interacts
with plasmid DNA probably involving electrostatic binding of cat-
ionic phosphonium salt to negatively charged plasmid DNA under
physiological conditions. This can induce damage and partial dena-
turation of supercoiled plasmid DNA and alter its native structure.
2.2. In vitro antibacterial activity
Antibacterial activity of the synthesized compounds was tested
on Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella
pneumoniae and Proteus spp. strains. Table 1 shows the MIC (min-
imum inhibitory concentrations) of the phosphonium salts in com-
parison with vancomycin as a reference drug.
All synthesized compounds did no exhibit antibacterial activity
against gram-negative bacteria. Phosphonium salts without ketal
protection group (4, 9, 12, 16, 19, 21, 25) did not have antibacterial
activity at all.
The quantity and relative position of pharmacophore phospho-
nium groups in pyridoxine derivatives had a significant effect on
their antibacterial activity. Monophosphonium salts 3 and 15 con-
taining a six-membered ketal cycle were almost inactive (MIC
P1000
lg/ml). Phosphonium salt 8, containing seven-membered
3. Conclusion
ketal cycle, was characterized by somewhat lower MIC of 100
lg/
ml. Bisphosphonium salt 18 containing methylenephosphonium
We have synthesized a series of novel phosphonium salts on the
basis of pyridoxine and 6-hydroxymethyl pyridoxine. 5,6-bis[tri-
phenylphosphonio(methyl)]-2,2,8-trimethyl-4H-[1,3]dioxino[4,5-
c]pyridine dichloride (compound 20) demonstrated high activity
against S. aureus and S. epidermidis with minimal inhibitory concen-
groups in the 2 and 5 positions of the pyridine ring was found to
be more effective with MIC value of 62.5 and 50 lg/ml for S. aureus
and S. epidermidis, respectively. Trisphosphonium salt 24 showed
similar activity to that of compound 18.
The maximum antibacterial effect was observed for bisphos-
phonium salt 20 containing methylenephosphonium groups in 5
tration of 5
lg/ml. Structure–activity relationships of phosphonium
HO
O
HO
HO
O
O
HO
a-g
O
j
OH
O
h
O
i
P+Ph3
P+Ph3
P+Ph3
P+Ph3
OH
OH
Cl
Cl
Ph3+P
Ph3+P
N
HO
N+
H
Cl
N
22
N
24
N
23
4Cl-
1
3Cl-
25
Scheme 7. Reagents and conditions: (a) (CH3)2CO, HCl, 0 °C, 24 h;32 (b) H2O, H2CO, NaOH, 70 °C, 60 h;33 (c) (CH3)2CO, 2,2-dimetoxypropane, HCl, rt, 30 h;35 (d) CH2Cl2, Ac2O,
Et3N, reflux, 30 h;36 (e) CHCl3, MCPBA, rt, 48 h;36 (f) Ac2O, 100 °C, 4 h;36 (g) C2H5OH, C2H5ONa, 0 °C, 2 h;36 (h) CHCl3, MsCl, Et3N, 0 °C to rt, 10 h; (i) PPh3, CH3CN, reflux, 7 h; (j)
H2O, HCl, 25 °C, 24 h.

M. V. Pugachev et al. / Bioorg. Med. Chem. 21 (2013) 4388–4395
4391
Table 1
In vitro antibacterial activities of phosphonium salts
Compound
MIC (lg/ml)
Gram-positive bacteria
Gram-negative bacteria
S. aureus
S. epidermidis
K. pneumoniae
Proteus spp.
3
4
8
9
12
15
16
18
19
20
21
24
1000
1000
100
1000
1000
>1000
>1000
62.5
250
5
1000
50
500
2.5
100
1000
100
1000
1000
>1000
>1000
50
250
5
1000
50
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
25
500
2.5
Vancomycin
Figure 2. Agarose gel electrophoresis of DNA from S. aureus treated with compound
20. Lanes: M1, M2 – DNA ladders; control – DNA from untreated cells; A–D – DNA
from cells treated with Compound 20 at 0.01, 0.1, 1.0, 10.0 mg/ml.
Table 2
Hydrodynamic diameter and zeta potential of plasmid DNA from S. aureus treated
with compound 20
Plasmid DNA
Hydrodynamic Polydispersity Zeta
diameter (nm) index potential (mV)
Control (untreated bacteria) 360.7 18.0
0.425
0.717
À50.7 1.0
À46.6 6.9
Bacteria treated with 0.01
mg/ml compound 20
500.9 53.6
Bacteria treated with 0.1
mg/ml compound 20
452.3 46.2
0.844
À35.2 7.9
and are uncorrected. For TLC analysis silica gel plates from Sorbfil
(Krasnodar, Russia) were used with UV light (254 nm/365 nm) or
iron (III) chloride as developing agent. Column chromatography
was performed on silica gel (60–200 mesh) from Acros. Gas chro-
matography/mass spectrometry conditions. Instrument—DFS
«Thermo Electron Corporation». Method of ionization—Electron
ionization. Electron energy—70 eV. Ion source temperature—
280 °C. Capillary column DB-5MS (30 Â 0.25). Gas-carrier—helium.
Gas-carrier rate—1 ml/min. Injector temperature—250 °C. Temper-
ature gradient: 50 °C (1 min)—6 grad/min—120 °C—20 grad/min—
Figure 1. (a) Microphotographs of S. aureus bacteria treated with compound 20.
Treated cells were captured in agarose gel, lysed, and stained with SYBR Green I. (K)
Untreated cells; (A–C) cells treated with compound 20 at 10, 1 and 0.01 mg/ml. (b)
Distribution area of DNA fragments.
salt derivatives revealed in our study are of interest in the develop-
ment of new antimicrobials.
280 °C (15 min). Sample volume—0.3 ll. MALDI-TOF experiments
were performed using an ULTRAFLEX III TOF/TOF mass spectrome-
ter (Bruker Daltonik GmbH, Germany). The spectra of positive ions
were recorded in linear mode with ion acceleration up to 20 keV.
The energy of the laser beam was attenuated down to 70% of the
full laser power. Analyte and p-nitroaniline matrix compounds
were dissolved in acetone (1% in case matrix and 0.1% in case ana-
lyte) using glassware. The samples were prepared by a «dried-
droplet» method: 0.3 ll droplets of the solutions were deposited
on the marked spots on the standard target plate «AnchorChip»
(Bruker Daltonik GmbH, Germany) and left to dry.
4. Experimental
4.1. General information
1H, 13C, 31P NMR spectra were recorded on a ‘Bruker AVANCE
400’ at operating frequency 400, 101.56, and 161.92 MHz respec-
tively. Chemical shifts were measured with reference to the resid-
ual protons of the solvents (DMSO-d6, 1H, 2.50 ppm, 13C,
39.52 ppm; CDCl3, 1H, 7.26 ppm, 13C, 77.16 ppm). Phosphoric acid
was used as an external standard for the 31P NMR. Coupling con-
stants (J) are given in Hertz (Hz). The following abbreviations are
used to describe coupling: s = singlet; d = doublet; t = triplet;
m = multiplet; dd = doublet of doublets; td = triplet of doublets;
br s = broad singlet. Melting points were determined using a Stan-
ford Research Systems MPA-100 OptiMelt melting point apparatus
4.1.1. Synthesis of hydroxy derivatives
4.1.1.1. 9-Acetoxy-6-(hydroxymethyl)-3,3,8-trimethyl-1,5-dihy-
dro-[1,3]dioxepino[5,6-c]pyridin (6).
To a solution of com-
pound 5 (630 mg, 2.64 mmol), triethylamine (0.47 ml, 3.37 mmol)
and acetic anhydride (0.25 ml, 2.65 mmol) in 30 ml of

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M. V. Pugachev et al. / Bioorg. Med. Chem. 21 (2013) 4388–4395
dichloromethane was stirred at room temperature for 30 h. Then
the solvent was evaporated under reduced pressure. The dry residue
was dissolved in chloroform (30 ml) and extracted with water
(2 Â 50 ml), and the combined organic layers were concentrated.
The product was purified by column chromatography [dichloro-
methane]. Yield 49% (360 mg); light yellow solid; mp 61 °C; 1H
NMR (CDCl3): d 1.45 (s, 6H, 2CH3), 2.33 (s, 3H, CH3), 2.34 (s, 3H,
CH3), 4.56 (s, 2H, CH2), 4.69 (s, 2H, CH2), 4.72 (s, 2H, CH2). 13C
NMR (CDCl3): d 18.78 (s, CH3), 20.42 (s, CH3C@O), 23.65 (s, 2CH3),
58.90 (s, CH2O), 58.92 (s, CH2O), 61.21 (s, CH2O), 102.77 (s,
C(CH3)2), 129.53 (s, CPyr), 140.94 (s, CPyr), 141.76 (s, CPyr), 147.60
(s, CPyr), 150.33 (s, CPyr), 168.34 (s, CC@O). MS (EI): [M]+ 281.
3.84 mmol) and triethylamine (4.75 ml, 23.1 mmol) and methane-
sulfonyl chloride (2.2 ml, 19.2 mmol). Yield 24% (280 mg); light
yellow solid; mp 110 °C; 1H NMR (CDCl3): d 1.58 (s, 6H, 2CH3),
4.59 (s, 2H, CH2O), 4.63 (s, 2H, CH2Cl), 4.74 (s, 2H, CH2Cl), 4.96 (s,
2H, CH2Cl). 13C NMR (CDCl3): d 24.67 (s, 2CH3), 37.25 (s, CH2Cl),
41.29 (s, CH2Cl), 44.48 (s, CH2Cl), 58.41 (s, CH2O), 100.81 (s,
C(CH3)2), 128.31 (s, CPyr), 128.39 (s, CPyr), 145.02 (s, CPyr), 145.83
(s, CPyr), 146.77 (s, CPyr). MS (EI): [M]+ 309.
4.1.3. General procedure for the preparation of phosphonium
salts
To a solution of the chloro derivative of pyridoxine (1 equiv) in
30 ml of acetonitrile were added triphenylphosphine (2–6 equiv).
The reaction mixture was refluxed for 7 h. Two different workup
procedures were used. For workup I, after refluxing the solvent
was evaporated under reduced pressure. The dry residue was dis-
solved in chloroform (30 ml) and extracted with water
(2 Â 100 ml), and the combined water layers were evaporated un-
der reduced pressure. The product was recrystallized from acetone.
For workup II, after refluxing the solvent was evaporated under re-
duced pressure. The dry residue was dissolved in water (30 ml) and
the precipitate was filtered. The filtrate was evaporated under re-
duced pressure. The product was recrystallized from acetone.
4.1.2. General procedure for the preparation of chloro
derivatives
To a solution of hydroxy derivatives (1 equiv) and triethylamine
(1.4 equiv) in 15 ml of chloroform methanesulfonyl chloride
(1.2 equiv) was added dropwise at 0 °C and the reaction mixture
was stirred at room temperature for 10 h. Then the solvent was
evaporated under reduced pressure. The product was extracted
with ethyl acetate (2 Â 10 ml) and the combined organic layers
were concentrated under reduced pressure. The product was puri-
fied by column chromatography [ethyl acetate].
4.1.2.1. 9-Acetoxy-6-(chloromethyl)-3,3,8-trimethyl-1,5-dihy-
4.1.3.1. 5-[(Triphenylphosphonio)methyl]-2,2,8-trimethyl-4H-
dro-[1,3]dioxepino[5,6-c]pyridine (7).
The reaction was car-
[1,3]dioxino[4,5-c]pyridin chloride (3).
The reaction was
ried out following the general procedure with compound 6
(700 mg, 2.49 mmol) and triethylamine (0.49 ml, 3.49 mmol) and
methanesulfonyl chloride (0.23 ml, 2.99 mmol). Yield 90%
(670 mg); light yellow solid; mp 117 °C; 1H NMR (CDCl3): d 1.48
(s, 6H, 2CH3), 2.33 (s, 3H, CH3), 2.34 (s, 3H, CH3), 4.60 (s, 2H,
CH2), 4.70 (s, 2H, CH2), 4.97 (s, 2H, CH2). 13C NMR (CDCl3): d
18.98 (s, CH3), 20.47 (s, CH3C@O), 23.68 (s, 2CH3), 45.13 (s, CH2Cl),
59.01 (s, CH2O), 60.42 (s, CH2O), 102.79 (s, C(CH3)2), 132.68 (s,
carried out following the general procedure with compound 2
(600 mg, 2.63 mmol) and triphenylphosphine (1379 mg,
5.26 mmol). The product was obtained following workup I proce-
dure. Yield 60% (774 mg); white solid; mp 211 °C (dec.); 1H NMR
7
(CDCl3): d 1.24 (s, 6H, 2CH3), 2.24 (d, 3H, JHP = 2.2 Hz, CH3), 4.33
2
(s, 2H, CH2O), 5.31 (d, 2H, JHP = À14.3 Hz, CH2P), 7.55–7.73 (m,
16H, PPh3+CH). 13C NMR (CDCl3): d 18.60 (s, CH3), 24.72 (s, 2CH3),
1
24.90 (d, JCP = 48.4 Hz, CH2P), 58.94 (s, CH2O), 99.91 (s, C(CH3)2),
1
C
Pyr), 141.24 (s, CPyr), 142.76 (s, CPyr), 149.19 (s, 2CPyr), 168.19 (s,
117.51 (d, J = 8.2 Hz, CPyr), 117.61 (d, JCP = 85.7 Hz, i-CAr), 127.50
CC@O). MS (EI): [M+H]+ 300.
(d, J = 5.7 Hz, CPyr), 130.44 (d, JCP = 12.5 Hz, m-CAr), 134.20 (d,
3
4
2JCP = 10.0 Hz, o-CAr), 135.33 (d, JCP = 2.9 Hz, p-CAr), 141.24 (d,
4.1.2.2. 3-(Chloromethyl)-5-hydroxy-2,4-bis(hydroxymethyl)-6-
J = 5.7 Hz, CPyr), 146.27 (d, J = 2.4 Hz, CPyr), 148.14 (d, J = 4.0 Hz, CPyr).
methylpyridinium chloride (11).
A suspension of compound
31P NMR {H} (CDCl3): d 24.86 (s). MALDI-MS: [M]+ 454.
10 (900 mg, 3.26 mmol) and concentrated HCl (1 ml) in 20 ml of
water was stirred at 55 °C for 72 h. Then the solvent was evapo-
rated under reduced pressure. The product was recrystallized from
ethanol. Yield 52% (430 mg); white solid; mp 157 °C (dec.); 1H
NMR (DMSO-d6): d 2.67 (s, 3H, CH3), 4.86 (s, 2H, CH2), 4.87 (s,
2H, CH2), 5.04 (s, 2H, CH2), 6.20 (br s, 2H, 2OH), 10.98 (br s, 1H,
OH). 13C NMR (DMSO-d6): d 14.94 (s, CH3), 37.01 (s, CH2Cl), 55.10
(s, CH2O), 57.27 (s, CH2O), 131.59 (s, CPyr), 143.05 (s, CPyr), 143.24
(s, CPyr), 144.71 (s, CPyr), 151.73 (s, CPyr). MALDI-MS: [MÀH]+ 217.
4.1.3.2. 6-[(Triphenylphosphonio)methyl]-9-acetoxy-3,3,8-tri-
methyl-1,5-dihydro-[1,3]dioxepino[5,6-c]pyridin
chloride
(8). The reaction was carried out following the general proce-
dure with compound 7 (500 mg, 1.66 mmol) and triphenylphos-
phine (873 mg, 3.33 mmol). The product was obtained following
workup II procedure. The product was recrystallized from ethyl
acetate: chloroform (10:1). Yield 59% (553 mg); white solid; mp
204 °C (dec.); 1H NMR (CDCl3): d 1.35 (s, 6H, 2CH3), 1.73 (s, 3H,
CH3), 2.20 (s, 3H, CH3C@O), 4.54 (s, 2H, CH2O), 5.18 (s, 2H,
CH2O), 5.71 (br s, 2H, CH2P), 7.50–7.83 v (15H, PPh3). 13C NMR
(CDCl3): d 17.31 (s, CH3), 20.28 (s, CH3C@O), 23.60 (s, 2CH3),
4.1.2.3. 5-(Chloromethyl)-3,3,9,9-tetramethyl-7,11-dihydro-1H-
[1,3]dioxepino[5,6-b][1,3]dioxino[5,4-d]pyridine (14).
The
1
reaction was carried out following the general procedure with
compound 13 (790 mg, 2.68 mmol) and triethylamine (0.52 ml,
3.75 mmol) and methanesulfonyl chloride (0.25 ml, 3.21 mmol).
The reaction mixture was refluxed for 5 h. The unstable product
was used immediately. Yield 84% (810 mg); yellow oil; 1H NMR
(CDCl3): d 1.50 (s, 6H, 2CH3), 1.55 (s, 6H, 2CH3), 4.62 (s, 4H,
2CH2), 4.65 (s, 2H, CH2), 4.73 (s, 2H, CH2), 4.90 (s, 2H, CH2). 13C
NMR (CDCl3): d 23.69 (s, 2CH3), 24.59 (s, 2CH3), 41.49 (s, CH2Cl),
58.18 (s, CH2O), 58.56 (s, CH2O), 66.39 (s, CH2O), 100.06 (s,
C(CH3)2), 102.49 (s, C(CH3)2), 125.12 (s, CPyr), 129.50 (s, CPyr),
142.00 (s, CPyr), 144.63 (s, CPyr), 148.87 (s, CPyr). MS (EI): [M]+ 313.
31.92 (d, JCP = 60.3 Hz, CH2P), 58.37 (s, CH2O), 61.59 (s, CH2O),
1
102.70 (s, C(CH3)2), 120.37 (d, JCP = 89.5 Hz, i-CAr), 129.70 (d,
2
3JCP = 12.7 Hz, m-CAr), 133.82 (d, JCP = 10.1 Hz, o-CAr), 134.03 (d,
4JCP = 2.5 Hz, p-CAr), 141.54 (s, CPyr), 141.85 (s, CPyr), 143.13 (d,
J = 7.2 Hz, CPyr), 147.15 (s, CPyr), 168.04 (s, CC@O). 31P NMR {H}
(CDCl3): d 23.80 (s). MALDI-MS: [M]+ 526.
4.1.3.3.
bis(hydroxymethyl)-2-methyl-pyridin-1-ium
(12). To a suspension of compound 11 (350 mg, 1.38 mmol)
5-[(Triphenylphosphonio)methyl]-3-hydroxy-4,6-
dichloride
in 30 ml of acetonitrile were added triphenylphosphine (725 mg,
2.77 mmol). The reaction mixture was refluxed for 7 h. The precip-
itate was filtered and then was washed with acetonitrile
(2 Â 10 ml). Yield 82% (585 mg); white solid; mp 222 °C (dec.);
1H NMR (DMSO-d6): d 2.67 (s, 3H, CH3), 4.02 (s, 2H, CH2O), 4.28
4.1.2.4.
no[4,5-c]pyridine (23).
ing the general procedure with compound 22 (980 mg,
5,6,8-Tris(chloromethyl)-2,2-dimethyl-4H-[1,3]dioxi-
The reaction was carried out follow-

M. V. Pugachev et al. / Bioorg. Med. Chem. 21 (2013) 4388–4395
4393
2
(s, 2H, CH2O), 5.62 (d, 2H, JHP = À14.8 Hz, CH2P), 6.23 (br s, 1H,
4.1.3.7. 5,6,8-Tris[(triphenylphosphonio)methyl]-2,2-dimethyl-
4H-[1,3]dioxino[4,5-c]pyridine trichloride (24). The reac-
OH), 7.68–7.93 (m, 15H, PPh3), 10.90 (br s, 1H, OH). 13C NMR
(DMSO-d6): d 15.17 (s, CH3), 22.92 (d, JCP = 49.7 Hz, CH2P), 55.40
(s, CH2O), 57.62 (s, CH2O), 117.50 (d, JCP = 85.6 Hz, i-CAr), 124.13
1
tion was carried out following the general procedure with com-
pound 23 (200 mg, 0.65 mmol) and triphenylphosphine
(1013 mg, 3.86 mmol). The product was obtained following work-
up I procedure. Yield 85% (601 mg); white solid; mp 193 °C (dec.);
1H NMR (CDCl3): d 0.79 (s, 6H, 2CH3), 3.97 (s, 2H, CH2O), 4.81 (br s,
2H, CH2P), 4.99 (d, 2H, 2JHP = À13.9 Hz, CH2P), 5.75 (br s, 2H, CH2P),
7.03–7.76 (m, 45H, 3PPh3). 13C NMR (CDCl3): d 23.98 (s, 2CH3),
1
3
(d, J = 8.0 Hz, CPyr), 130.20 (d, JCP = 12.6 Hz, m-CAr), 134.36 (d,
2JCP = 10.1 Hz, o-CAr), 135.34 (d, JCP = 1.5 Hz, p-CAr), 143.05 (d,
4
J = 2.2 Hz, CPyr), 144.81 (s, CPyr), 145.37 (d, J = 5.0 Hz, CPyr), 151.20
(d, J = 1.9 Hz, CPyr). 31P NMR {H} (DMSO-d6): d 22.80 (s). MALDI-
MS: [MÀH]+ 444.
1
1
26.30 (d, JCP = 46.8 Hz, CH2P), 28.90 (d, JCP = 49.6 Hz, CH2P),
1
4.1.3.4.
methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridin
(15). The reaction was carried out following the general pro-
8-[(Triphenylphosphonio)methyl]-5,6-bis(hydroxy-
31.88 (d, JCP = 59.0 Hz, CH2P), 58.50 (s, CH2O), 101.36 (s,
1
1
chloride
C(CH3)2), 116.78 (d, JCP = 84.7 Hz, i-CAr), 117.91 (d, JCP = 86.4 Hz,
i-CAr), 118.89 (d, JCP = 88.8 Hz, i-CAr), 122.10 (td, J = 8.9, 3.0 Hz,
CPyr), 130.13 (d, JCP = 12.7 Hz, m-CAr), 130.16 (br s, CPyr), 130.20
(d, JCP = 12.6 Hz, m-CAr), 130.63 (d, JCP = 12.6 Hz, m-CAr), 133.21
1
3
cedure with compound 14 (600 mg, 1.91 mmol) and triphenyl-
phosphine (1001 mg, 3.82 mmol). The product was obtained
following workup II procedure. Yield 58% (594 mg); light yellow
solid; mp 202 °C (dec.); 1H NMR (DMSO-d6): d 1.39 (s, 6H, 2CH3),
4.22 (s, 2H, CH2O), 4.42 (s, 2H, CH2O), 4.83 (br s, 1H, OH), 4.96 (s,
3
3
2
2
(d, JCP = 10.0 Hz, o-CAr), 133.82 (d, JCP = 10.3 Hz, o-CAr), 134.34
2
4
(d, JCP = 9.6 Hz, o-CAr), 134.40 (d, JCP = 3.0 Hz, p-CAr), 135.21 (d,
4JCP = 1.3 Hz, p-CAr), 135.59 (d, JCP = 1.7 Hz, p-CAr), 138.93 (dd,
4
2H, CH2O), 5.23 (d, 2H, JHP = À14.8 Hz, CH2P), 5.32 (br s, 1H, OH),
J = 7.6, 3.9 Hz, CPyr), 141.76 (t, J = 5.2 Hz, CPyr), 146.29 (t,
2
7.68–7.85 (m, 15H, PPh3). 13C NMR (DMSO-d6): d 24.23 (s, CH3),
J = 2.9 Hz, CPyr). 31P NMR {H} (CDCl3): d 18.44 (d, JPP = 7.7 Hz),
7
1
7
25.57 (d, JCP = 54.8 Hz, CH2P), 55.43 (s, CH2O), 58.47 (s, CH2O),
62.01 (s, CH2O), 100.10 (s, C(CH3)2), 119.40 (d, JCP = 87.5 Hz, i-
CAr), 128.46 (s, CPyr), 129.76 (d, JCP = 12.7 Hz, m-CAr), 131.12 (d,
19.87 (d, JPP = 7.7 Hz), 24.71 (s). MALDI-MS: [MÀ3H]+ 988.
1
3
4.1.4. General procedure of removing the ketal protecting group
of phosphonium salts
A mixture of phosphonium salt (1 equiv) and concentrated HCl
(1 ml) in 20 ml of water was stirred at 25 °C for 24 h. After stirring
the solvent was evaporated under reduced pressure.
2
J = 1.5 Hz,
C
Pyr), 133.87 (d, JCP = 10.3 Hz, o-CAr), 134.49 (d,
4JCP = 2.1 Hz, p-CAr), 135.17 (d, J = 7.8 Hz, CPyr), 145.51 (d,
J = 7.0 Hz, CPyr), 148.49 (s, CPyr). 31P NMR {H} (DMSO-d6): d 23.53
(s). MALDI-MS: [M]+ 500.
4.1.3.5.
5,8-Bis[(triphenylphosphonio)methyl]-2,2-dimethyl-
The reaction
4.1.4.1.
(hydroxymethyl)-2-methylpyridin-1-ium
(4). The general procedure was followed using compound 3
5-[(Triphenylphosphonio)methyl]-3-hydroxy-4-
4H-[1,3]dioxino[4,5-c]pyridine dichloride (18).
dichloride
was carried out following the general procedure with compound
17 (400 mg, 1.53 mmol) and triphenylphosphine (1600 mg,
6.11 mmol). The product was obtained following workup I proce-
dure. Yield 88% (1057 mg); white solid; mp 206 °C (dec.); 1H
NMR (CDCl3): d 1.02 (s, 6H, 2CH3), 4.07 (s, 2H, CH2O), 5.19 (d,
(250 mg, 0.42 mmol). Yield quantitative (247 mg); white solid;
7
mp 231 °C (dec.); 1H NMR (DMSO-d6): d 2.57 (d, 3H, JHP = 1.5 Hz,
2
CH3), 3.87 (s, 2H, CH2O), 5.58 (d, 2H, JHP = À14.7 Hz, CH2P),
7.69–7.95 (m, 16H, PPh3+CH), 10.89 (br s, 1H, OH). 13C NMR
2
2
1
2H, JHP = À14.3 Hz, CH2P), 5.28 (d, 2H, JHP = À14.6 Hz, CH2P),
(DMSO-d6): d 15.40 (s, CH3), 23.67 (d, JCP = 48.9 Hz, CH2P), 54.92
7.42–7.70 (m, 31H, 2PPh3+CH). 13C NMR (CDCl3): d 24.44 (s, CH3),
(s, CH2O), 116.85 (d, 1JCP = 85.9 Hz, i-CAr), 124.64 (d, J = 7.7 Hz, CPyr),
1
1
3
25.30 (d, JCP = 48.5 Hz, CH2P), 27.24 (d, JCP = 53.2 Hz, CH2P),
130.31 (d, JCP = 12.6 Hz, m-CAr), 132.97(s, CPyr), 134.26 (d,
1
58.32 (s, CH2O), 101.02 (s, C(CH3)2), 117.01 (d, JCP = 85.6 Hz, i-
2JCP = 10.2 Hz, o-CAr), 135.44 (d, 4JCP = 2.1 Hz, p-CAr), 143.40 (s, CPyr),
143.59 (s, CPyr), 152.31 (d, J = 2.8 Hz, CPyr). 31P NMR {H} (DMSO-d6):
d 23.98 (s). MALDI-MS: [M]+ 415.
C
Ar), 118.61 (d, 1JCP = 87.2 Hz, i-CAr), 120.26 (dd, J = 7.9, 2.2 Hz, CPyr),
3
128.77 (d, J = 5.5 Hz, CPyr), 129.96 (d, JCP = 12.9 Hz, m-CAr), 130.59
(d, 3JCP = 12.6 Hz, m-CAr), 133.80 (d, 2JCP = 10.2 Hz, o-CAr), 134.00 (d,
2JCP = 10.0 Hz, o-CAr), 134.69 (d, JCP = 2.8 Hz, p-CAr), 135.35 (d,
4.1.4.2.
bis(hydroxymethyl)-2-methylpyridin-1-ium
(9). The reaction was carried out following the general proce-
6-[(Triphenylphosphonio)methyl]-3-hydroxy-4,5-
4
4JCP = 2.6 Hz, p-CAr), 138.56 (dd, J = 8.0, 4.1 Hz, CPyr), 141.71 (dd,
dichloride
J = 5.0, 1.1 Hz, CPyr). 146.83 (dd, J = 6.3, 2.7 Hz, CPyr). 31P NMR {H}
7
7
(CDCl3): d 24.63 (d, JPP = 5.9 Hz), 25.96 (d, JPP = 5.9 Hz). MALDI-
MS: [MÀH]+ 714.
dure with compound 8 (400 mg, 0.71 mmol). Yield quantitative
(394 mg); white solid; mp 85 °C (dec.); 1H NMR (DMSO-d6): d
1.91 (s, 3H, CH3), 4.50 (s, 2H, CH2O), 4.72 (s, 2H, CH2O), 5.71 (d,
2
4.1.3.6. 5,6-Bis[(triphenylphosphonio)methyl]-2,2,8-trimethyl-
4H-[1,3]dioxino[4,5-c]pyridine dichloride (20).
was carried out following the general procedure with compound
10 (400 mg, 1.45 mmol) and triphenylphosphine (1519 mg,
5.80 mmol). The product was obtained following workup I proce-
dure. Yield 85% (986 mg); white solid; mp 218 °C (dec.); 1H NMR
2H, JHP = À14.3 Hz, CH2P), 5.80 (br s, 2H, 2OH), 7.65–7.86 (m,
The reaction
15H, PPh3). 13C NMR (DMSO-d6): d 17.03 (s, CH3), 29.06 (d,
1JCP = 59.1 Hz, CH2P), 55.79 (s, CH2O), 56.02 (s, CH2O), 120.45 (d,
3
1JCP = 86.8 Hz, i-CAr), 129.73 (d, JCP = 12.6 Hz, m-CAr), 133.76 (d,
4
2JCP = 10.1 Hz, o-CAr), 134.24 (d, JCP = 1.0 Hz, p-CAr), 136.25 (CPyr),
137.68 (CPyr), 144.07 (2CPyr), 149.78 (CPyr). 31P NMR {H} (DMSO-
7
(CDCl3): d 1.12 (s, 6H, 2CH3), 1.81 (d, 3H, JHP = 2.4 Hz, CH3), 3.94
d6): d 23.53 (s). MALDI-MS: [MÀH]+ 444.
(s, 2H, CH2O), 5.06 (br s, 2H, CH2P), 5.62 (br s, 2H, CH2P), 7.57–
7.72 (m, 30H, 2PPh3). 13C NMR (CDCl3): d 16.97 (s, CH3), 24.43 (s,
4.1.4.3.
2-[(Triphenylphosphonio)methyl]-3-hydroxy-4,5,6-
The
1
1
2CH3), 25.96 (d, JCP = 47.6 Hz, CH2P), 32.80 (d, JCP = 61.7 Hz,
tris(hydroxymethyl)pyridin-1-ium dichloride (16).
CH2P), 58.67 (s, CH2O), 100.08 (s, C(CH3)2), 117.08 (d, 1JCP = 84.7 Hz,
general procedure was followed using compound 15 (250 mg,
0.47 mmol). Yield quantitative (248 mg); light yellow solid; mp
163 °C (dec.); 1H NMR (DMSO-d6): d 4.29 (s, 2H, CH2O), 4.51 (s,
1
i-CAr), 118.21 (t, J = 8.6 Hz, CPyr), 119.97 (d, JCP = 89.9 Hz, i-CAr),
128.09 (d, J = 4.5 Hz,
130.98 (d, JCP = 12.6 Hz, m-CAr), 133.69 (d, JCP = 10.2 Hz, o-CAr),
134.34 (d, JCP = 10.0 Hz, o-CAr), 134.35 (d, JCP = 3.1 Hz, p-CAr),
3
C
Pyr), 129.89 (d, JCP = 12.9 Hz, m-CAr),
3
2
2
2H, CH2O), 4.73 (s, 2H, CH2O), 5.49 (d, 2H, JHP = À14.7 Hz, CH2P),
2
4
5.98 (br s, 3H, 3OH), 7.66–7.84 (m, 15H, PPh3), 10.37 (br s, 1H,
4
1
135.88 (d, JCP = 2.8 Hz, p-CAr), 140.14 (t, J = 6.3 Hz, CPyr), 145.55
OH). 13C NMR (DMSO-d6): d 26.38 (d, JCP = 55.7 Hz, CH2P), 55.43
(d, J = 2.8 Hz, CPyr), 146.99 (d, J = 4.0 Hz, CPyr). 31P NMR {H} (CDCl3):
d 20.91 (s), 26.42 (s). MALDI-MS: [MÀ2H]+ 728.
(s, CH2O), 55.81 (s, CH2O), 60.96 (s, CH2O), 119.43 (d, 1JCP = 87.6 Hz,
3
2
i-CAr), 129.80 (d, JCP = 12.7 Hz, m-CAr), 133.86 (d, JCP = 10.3 Hz, o-

4394
M. V. Pugachev et al. / Bioorg. Med. Chem. 21 (2013) 4388–4395
4
CAr), 134.53 (d, JCP = 2.8 Hz, p-CAr), 135.09 (d, J = 7.3 Hz, CPyr),
137.89 (s, CPyr), 148.37 (s, 2CPyr), 150.80 (d, J = 7.0 Hz, CPyr). 31P
NMR {H} (DMSO-d6): d 25.67 (s). MALDI-MS: [MÀH]+ 460.
(Kazan, Russia). Stock solutions of synthesized phosphonium salts
were prepared and sterilized with the use of 0.2 lm filter.
Bacterial inoculum (5 Â 105 CFU/ml) was cultured in Müller-
Hinton broth (pH 7.3) in the presence of phosphonium salts at dif-
ferent concentrations at 35 °C for 18–24 h. Minimum inhibitory
concentrations were determined as the lowest ones at which no
growth of bacteria was detected.
4.1.4.4.
2,5-Bis[(triphenylphosphonio)methyl]-3-hydroxy-4-
The gen-
(hydroxymethyl)pyridine-1-ium trichloride (19).
eral procedure was followed using compound 18 (250 mg,
0.32 mmol). The product was precipitated from water and filtered
off. Yield 82% (204 mg); white solid; mp 236 °C (dec.); 1H NMR
4.3. Cytotoxic activity
2
(DMSO-d6): d 3.77 (s, 2H, CH2O), 5.27 (d, 2H, JHP = À14.9 Hz,
2
CH2P), 5.56 (d, 2H, JHP = À12.9 Hz, CH2P), 5.83 (br s, 1H, OH),
The human skin fibroblasts were cultured in DMEM supple-
mented with 10% FBS, 2 mM L-glutamine, 100 lg/ml penicillin
and 100 U/ml streptomycin. The cells were seeded in 96-well
plates at the density of 3000 cells per well and allowed to attach
overnight. Cells were cultured in the presence of phosphonium salt
7.28–7.93 (m, 31H, 2PPh3+CH), 10.21 (br s, 1H, OH). 13C NMR
(DMSO-d6):
d
23.53 (d, 1JCP = 47.9 Hz, CH2P), 27.52 (d,
1JCP = 56.0 Hz, CH2P), 54.33 (s, CH2O), 117.10 (d, JCP = 85.8 Hz, i-
1
1
C
Ar), 119.95 (d, JCP = 88.4 Hz, i-CAr), 123.18 (d, J = 8.3 Hz, CPyr),
3
3
129.59 (d, JCP = 12.7 Hz, m-CAr), 130.13 (d, JCP = 12.4 Hz, m-CAr),
133.61 (d, JCP = 10.2 Hz, o-CAr), 133.97 (d, JCP = 9.9 Hz, o-CAr),
134.29 (d, JCP = 2.0 Hz, p-CAr), 135.21 (d, JCP = 2.1 Hz, p-CAr),
137.86 (d, J = 5.3 Hz, CPyr), 139.82 (dd, J = 6.7, 4.3 Hz, CPyr), 141.10
(d, J = 5.4 Hz, CPyr), 150.53 (dd, J = 7.7, 2.9 Hz, CPyr). 31P NMR {H}
solutions (1 lg/ml–1 mg/ml) for 72 h at 37 °C and 5% CO2. Grown
2
2
cells were washed with fresh medium and subjected to conven-
tional MTT assay. The colored product of MTT reduction by viable
cells was detected on Infinite 200 PRO analyzer at 550 nm (TECAN).
4
4
7
7
(DMSO-d6): d 23.79 (d, JPP = 3.1 Hz), 23.98 (d, JPP = 3.1 Hz). MAL-
4.4. DNA damage activity
DI-MS: [MÀ2H]+ 674.
4.4.1. Bacterial strain
4.1.4.5.
(hydroxymethyl)-6-methylpyridine-1-ium
(21). The general procedure was followed using compound
2,3-Bis[(triphenylphosphonio)methyl]-5-hydroxy-4-
S. aureus strain 25923 was obtained from National Center for
Prevention and Control of AIDS and Infectious Diseases of the Min-
istry of Health of the Republic of Tatarstan. Bacteria were pre-
grown on blood agar plates at 37 °C. The colonies were detected
with the use of S. aureus Bio-Rad Pastorex Staph Plus kit. Bacteria
were cultured in Müller-Hinton broth (pH 7.3) at 37 °C. Night cul-
ture was centrifuged at 10,000 rpm for 10 min and resuspended in
sterile phosphate buffer saline (PBS). Bacterial suspension
(5 Â 105 CFU/ml) was pretreated for DNA damage measurement
with compound 20 at different concentration (0.1; 1 and 10 mg/
ml) for 1 h at 37 °C.
trichloride
20 (250 mg, 0.31 mmol). Yield quantitative (249 mg); white solid;
7
mp 116 °C (dec.); 1H NMR (DMSO-d6): d 1.87 (d, 3H, JHP = 2.2 Hz,
CH3), 3.93 (s, 2H, CH2O), 4.98 (br s, 2H, CH2P), 5.67 (d, 2H,
2JHP = À14.3 Hz, CH2P), 7.57–7.87 (m, 30H, 2PPh3), 9.42 (br s, 1H,
OH). 13C NMR (DMSO-d6): d 17.94 (s, CH3), 23.82 (d, J = 47.1 Hz,
CH2P), 31.32 (d, J = 61.0 Hz, CH2P), 54.91 (s, CH2O), 117.88 (d,
1
1JCP = 84.6 Hz, i-CAr), 120.48 (d, JCP = 89.4 Hz, i-CAr), 121.85 (t,
3
J = 8.5 Hz,
C
Pyr), 129.66 (d, JCP = 12.7 Hz, m-CAr), 130.27 (d,
3JCP = 12.4 Hz, m-CAr), 133.39 (d, JCP = 10.1 Hz, o-CAr), 134.18 (d,
2
4JCP = 2.0 Hz, p-CAr), 134.23 (d, JCP = 10.0 Hz, o-CAr), 135.32 (d,
4.4.2. In situ detection of DNA damage
2
4JCP = 2.0 Hz, p-CAr), 137.35 (d, J = 4.7 Hz, CPyr), 139.60 (t,
J = 6.2 Hz, CPyr), 145.76 (d, J = 3.8 Hz, CPyr), 148.81 (d, J = 2.9 Hz,
Cells were centrifuged at 3000 g for 15 min and washed twice
with PBS after pretreatment with compound 20. To confirm the
presence of DNA breakage in cells with diffused DNA spots, the
DNA breakage detection-fluorescence in situ hybridization (DBD-
FISH) procedure was used The cells were immersed in agarose
microgels and were lysed as described above.38 The lysed cells cap-
tured in microgel were stained with SYBRGreen1 and analyzed un-
der AxioScope A1 fluorescence microscope (Carl Zeiss). DNA-
damaging activity was assessed by detecting distribution area of
DNA fragments from fluorescent microphotographs using panels
of contrast colors in Corel Draw 5X. The statistical significance of
the changes in DNA-damaging activity was revealed with Dun-
nett’s test.
C
Pyr). 31P NMR {H} (CDCl3): d 21.27 (s), 23.74 (s). MALDI-MS:
[MÀ3H]+ 687.
4.1.4.6. 2,3,6-Tris[(triphenylphosphonio)methyl]-5-hydroxy-4-
(hydroxymethyl)pyridine-1-ium tetrachloride (25).
The
general procedure was followed using compound 24 (200 mg,
0.18 mmol). Yield quantitative (199 mg); light yellow solid; mp
205 °C (dec.); 1H NMR (CDCl3): d 3.84 (s, 2H, CH2O), 4.62 (br s,
2H, CH2P), 5.06 (br s, 2H, CH2P), 5.66 (br s, 2H, CH2P), 7.19–7.66
(m, 45H, 3PPh3), 9.90 (br s, 1H, OH). 13C NMR (CDCl3): d 26.94 (d,
1
1JCP = 48.3 Hz, CH2P), 30.63 (d, JCP = 49.2 Hz, CH2P), 32.43 (d,
1
1JCP = 59.4 Hz, CH2P), 56.49 (s, CH2O), 117.61 (d, JCP = 85.1 Hz, i-
1
1
C
C
C
C
C
Ar), 118.19 (d, JCP = 86.2 Hz, i-CAr), 119.50 (d, JCP = 88.9 Hz, i-
4.4.3. DNA electrophoresis
3
Ar), 126.14 (td, J = 8.9, 3.0 Hz, CPyr), 130.24 (d, JCP = 12.6 Hz, m-
Total DNA from S. aureus was extracted as described earlier.39
Agarose gele electrophoresis was carried out in 1% gele at 100 V
for 45 min in TBE buffer (90 mM Tris–HCl, 2 mM EDTA, 90 mM bo-
3
3
Ar), 130.28 (d, JCP = 12.7 Hz, m-CAr), 130.62 (d, JCP = 12.6 Hz, m-
2
2
Ar), 133.71 (d, JCP = 10.8 Hz, o-CAr), 133.81 (d, JCP = 10.4 Hz, o-
4
2
Ar), 134.45 (d, JCP = 2.8 Hz, p-CAr), 134.46 (d, JCP = 9.3 Hz, o-CAr),
ric acid, pH 8.0). DNA was stained with 1.5 lg/ml ethidium bro-
mide and detected on Chemi-Doc XRS, Bio-Rad).
4
4
135.08 (d, JCP = 1.8 Hz, p-CAr), 135.52 (d, JCP = 1.7 Hz, p-CAr),
138.85 (d, J = 3.9 Hz, CPyr), 139.99 (dd, J = 7.6, 3.4 Hz, CPyr), 141.30
(t, J = 5.7 Hz, CPyr), 150.82 (t, J = 2.9 Hz, CPyr). 31P NMR {H} (CDCl3):
4.4.4. pDNA extraction and pDNA hydrodynamic diameter and
zeta-potential measurements
7
7
d 20.55 (d, JPP = 8.1 Hz), 22.28 (d, JPP = 8.1 Hz), 23.84 (s). MALDI-
MS: [MÀ4H]+ 946.
Plasmid DNA was isolated using GeneJET Plasmid Miniprep Kit
(Thermo Scientific). Hydrodynamic diameter and zeta potential of
plasmid DNA were analyzed by dynamic light scattering technique
on a Zetasizer Nano ZS instrument (Malvern instruments, UK).
Plasmid DNA was dissolved in HEPES buffer (pH 7.4) at the concen-
tration of 0.1 mg/ml. The measurements were carried out in
triplicates.
4.2. Antibacterial activity
Antibacterial activity was tested on clinical strains of S. aureus,
S. epidermidis, K. pneumoniae and Proteus spp. Bacteria were pro-
vided from the Kazan Institute of Epidemiology and Microbiology

M. V. Pugachev et al. / Bioorg. Med. Chem. 21 (2013) 4388–4395
4395
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